Isolation of a highly quiescent subpopulation of primitive leukemic cells in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is characterized by an increased proliferative activity of the leukemic progenitors that produce an elevated number of mature granulocytes. Nevertheless, cell cycle-active agents, even in very high doses, are alone unable to eradicate the leukemic clone, suggesting the presence of a rare subset of quiescent leukemic stem cells. To isolate such cells, we first used Hoechst 33342 and Pyronin Y staining to obtain viable G(0) and G(1)/S/G(2)/M fractions of CD34(+) cells by fluorescence-activated cell sorting (FACS) from 6 chronic-phase CML patients' samples and confirmed the quiescent and cycling status of the 2 fractions by demonstration of expected patterns of Ki-67 and D cyclin expression. Leukemic (Ph(+)/BCR-ABL(+)) cells with in vitro progenitor activity and capable of engrafting immunodeficient mice were identified in the directly isolated G(0) cells. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that many leukemic CD34(+) G(0) cells also expressed BCR-ABL mRNA. CD34(+) from 8 CML patients were also labeled with carboxyfluorescein diacetate succinimidyl diester (CFSE) before being cultured (with and without added growth factors) to allow viable cells that had remained quiescent (ie, CFSE(+)) after 4 days to be retrieved by FACS. Leukemic progenitors were again detected in all quiescent populations isolated by this second strategy, including those exposed to a combination of flt3-ligand, Steel factor, interleukin-3, interleukin-6, and granulocyte colony-stimulating factor. These findings provide the first direct and definitive evidence of a deeply but reversibly quiescent subpopulation of leukemic cells in patients with CML with both in vitro and in vivo stem cell properties.